Plants and seeds of high oil corn variety hoi002

ABSTRACT

According to the invention, there is provided seed and plants of the corn variety designated HOI002. This invention thus relates to the plants, seeds and tissue cultures of the variety HOI002, and to methods for producing a corn plant produced by crossing a corn plant of variety HOI002 with itself or with another corn plant, such as a plant of another variety. This invention further relates to corn seeds and plants produced by crossing plants of variety HOI002 with plants of another variety, such as another inbred line, and to crosses with related species. This invention further relates to the inbred and hybrid genetic complements of plants of variety HOI002, and also to the SSR and isozyme typing profiles of corn variety HOI002.

This application claims the priority of U.S. Provisional PatentApplication Ser. No. 60/343,964, filed Dec. 28, 2001, the entiredisclosure of which is specifically incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of corn breeding.In particular, the invention relates to inbred corn seed and plants ofthe variety designated HOI002, and tissue cultures thereof, as well asmethods of use thereof

2. Description of Related Art

The goal of field crop breeding is to combine various desirable traitsin a single variety/hybrid. Such desirable traits include greater yield,better stalks, better roots, resistance to insecticides, herbicides,pests, and disease, tolerance to heat and drought, reduced time to cropmaturity, better agronomic quality, higher nutritional value, anduniformity in germination times, stand establishment, growth rate,maturity, and fruit size.

Breeding techniques take advantage of a plant's method of pollination.There are two general methods of pollination: a plant self-pollinates ifpollen from one flower is transferred to the same or another flower ofthe same plant. A plant cross-pollinates if pollen comes to it from aflower on a different plant.

Corn plants (Zea mays L.) can be bred by both self-pollination andcross-pollination. Both types of pollination involve the corn plant'sflowers. Corn has separate male and female flowers on the same plant,located on the tassel and the ear, respectively. Natural pollinationoccurs in corn when wind blows pollen from the tassels to the silks thatprotrude from the tops of the ear shoot.

Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants produces a uniform population ofhybrid plants that are heterozygous for many gene loci. Conversely, across of two plants each heterozygous at a number of loci produces apopulation of hybrid plants that differ genetically and are not uniform.The resulting non-uniformity makes performance unpredictable.

The development of uniform corn plant hybrids requires the developmentof homozygous inbred plants, the crossing of these inbred plants, andthe evaluation of the crosses. Pedigree breeding and recurrent selectionare examples of breeding methods used to develop inbred plants frombreeding populations. Those breeding methods combine the geneticbackgrounds from two or more inbred plants or various other broad-basedsources into breeding pools from which new inbred plants are developedby selfing and selection of desired phenotypes. The new inbreds arecrossed with other inbred plants and the hybrids from these crosses areevaluated to determine which of those have commercial potential.

The pedigree breeding method involves crossing two genotypes. Eachgenotype can have one or more desirable characteristics lacking in theother; or, each genotype can complement the other. If the two originalparental genotypes do not provide all of the desired characteristics,other genotypes can be included in the breeding population. Superiorplants that are the products of these crosses are selfed and selected insuccessive generations. Each succeeding generation becomes morehomogeneous as a result of self-pollination and selection. Typically,this method of breeding involves five or more generations of selfing andselection: S₁→S₂; S₂→S₃; S₃→S₄; S₄→S₅, etc. After at least fivegenerations, the inbred plant is considered genetically pure.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or non-inbredsource to an inbred that lacks that trait. This can be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriate locusor loci for the trait in question. The progeny of this cross are thenmated back to the superior recurrent parent (A) followed by selection inthe resultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny are heterozygous for locicontrolling the characteristic being transferred, but are like thesuperior parent for most or almost all other loci. The last backcrossgeneration would be selfed to give pure breeding progeny for the traitbeing transferred.

A single cross hybrid corn variety is the cross of two inbred plants,each of which has a genotype that complements the genotype of the other.The hybrid progeny of the first generation is designated F₁. Typically,F₁ hybrids are more vigorous than their inbred parents. This hybridvigor, or heterosis, is manifested in many polygenic traits, includingmarkedly improved yields, better stalks, better roots, better uniformityand better insect and disease resistance. In the development of hybridsonly the F₁ hybrid plants are typically sought. An F₁ single crosshybrid is produced when two inbred plants are crossed. A double crosshybrid is produced from four inbred plants crossed in pairs (A×B andC×D) and then the two F₁ hybrids are crossed again (A×B)×(C×D).

The development of a hybrid corn variety involves three steps: (1) theselection of plants from various germplasm pools; (2) the selfing of theselected plants for several generations to produce a series of inbredplants, which, although different from each other, each breed true andare highly uniform; and (3) crossing the selected inbred plants withunrelated inbred plants to produce the hybrid progeny (F₁). During theinbreeding process in corn, the vigor of the plants decreases. Vigor isrestored when two unrelated inbred plants are crossed to produce thehybrid progeny (F₁). An important consequence of the homozygosity andhomogeneity of the inbred plants is that the hybrid between any twoinbreds is always the same. Once the inbreds that give a superior hybridhave been identified, hybrid seed can be reproduced indefinitely as longas the homogeneity of the inbred parents is maintained. Conversely, muchof the hybrid vigor exhibited by F₁ hybrids is lost in the nextgeneration (F₂). Consequently, seed from hybrid varieties is not usedfor planting stock. It is not generally beneficial for farmers to saveseed of F₁ hybrids. Rather, farmers purchase F₁ hybrid seed for plantingevery year.

North American farmers plant tens of millions of acres of corn at thepresent time and there are extensive national and internationalcommercial corn breeding programs. A continuing goal of these cornbreeding programs is to develop corn hybrids that are based on stableinbred plants and have one or more desirable characteristics. Toaccomplish this goal, the corn breeder must select and develop superiorinbred parental plants.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a corn plant of thevariety designated HOI002. Also provided are corn plants having all thephysiological and morphological characteristics of the inbred cornvariety HIO002. The inbred corn plant of the invention may furthercomprise, or have, a cytoplasmic or nuclear factor that is capable ofconferring male sterility or otherwise preventing self-pollination, suchas by self-incompatibility. Parts of the corn plant of the presentinvention are also provided, for example, pollen obtained from an inbredplant and an ovule of the inbred plant.

The invention also concerns seed of the inbred corn variety HOI002. Asample of this seed has been deposited under ATCC Accession No.PTA-3788. The inbred corn seed of the invention may be provided as anessentially homogeneous population of inbred corn seed of the varietydesignated HOI002. Essentially homogeneous populations of inbred seedare those that consist essentially of the particular inbred seed, andare generally free from substantial numbers of other seed, so that theinbred seed forms between about 90% and about 100% of the total seed,and preferably, between about 95% and about 100% of the total seed. Mostpreferably, an essentially homogeneous population of inbred corn seedwill contain between about 98.5%, 99%, 99.5% and about 99.9% of inbredseed, as measured by seed grow outs. This corresponds to currentcommercial practice among the leading companies in the seed industry.

Therefore, in the practice of the present invention, inbred seedgenerally forms at least about 97% of the total seed. However, even if apopulation of inbred corn seed was found, for some reason, to containabout 50%, or even about 20% or 15% of inbred seed, this would still bedistinguished from the small fraction (generally less than 2% andpreferably less than 1%) of inbred seed that may be found within apopulation of hybrid seed, e.g., within a commercial bag of hybrid seed.In such a bag of hybrid seed offered for sale, Federal regulationsrequire that the hybrid seed be at least about 95% of the total seed, orbe labeled as a mixture. In the most preferred practice of theinvention, the female inbred seed that may be found within a bag ofhybrid seed will be about 1% of the total seed, or less, and the maleinbred seed that may be found within a bag of hybrid seed will benegligible, i.e., will be on the order of about a maximum of 1 per100,000, and usually less than this value.

The population of inbred corn seed of the invention can further beparticularly defined as being essentially free from hybrid seed. Theinbred seed population may be separately grown to provide an essentiallyhomogeneous population of inbred corn plants designated HOI002.

In another aspect of the invention, single locus converted plants ofvariety HOI002 are provided. The single transferred locus may preferablybe a dominant or recessive allele. Preferably, the single transferredlocus will confer such traits as male sterility, yield stability, waxystarch, yield enhancement, industrial usage, herbicide resistance,insect resistance, resistance to bacterial, fungal, nematode or viraldisease, male fertility, and enhanced nutritional quality. The singlelocus may be a naturally occurring maize gene introduced into the genomeof the variety by backcrossing, a natural or induced mutation, or atransgene introduced through genetic transformation techniques. Whenintroduced through transformation, a single locus may comprise one ormore transgenes integrated at a single chromosomal location.

In yet another aspect of the invention, an inbred corn plant of thevariety designated HOI002 is provided, wherein acytoplasmically-inherited trait has been introduced into said inbredplant. Such cytoplasmically-inherited traits are passed to progenythrough the female parent in a particular cross. An exemplarycytoplasmically-inherited trait is the male sterility trait.Cytoplasmic-male sterility (CMS) is a pollen abortion phenomenondetermined by the interaction between the genes in the cytoplasm and thenucleus. Alteration in the mitochondrial genome and the lack of restorergenes in the nucleus will lead to pollen abortion. With either a normalcytoplasm or. the presence of restorer gene(s) in the nucleus, the plantwill produce pollen normally. A CMS plant can be pollinated by amaintainer version of the same variety, which has a normal cytoplasm butlacks the restorer gene(s) in the nucleus, and continue to be malesterile in the next generation. The male fertility of a CMS plant can berestored by a restorer version of the same variety, which must have therestorer gene(s) in the nucleus. With the restorer gene(s) in thenucleus, the offspring of the male-sterile plant can produce normalpollen grains and propagate. A cytoplasmically inherited trait may be anaturally occurring maize trait or a trait introduced through genetictransformation techniques.

In another aspect of the invention, a tissue culture of regenerablecells of a plant of variety HOI002 is provided. The tissue culture willpreferably be capable of regenerating plants capable of expressing allof the physiological and morphological characteristics of the variety,and of regenerating plants having substantially the same genotype asother plants of the variety. Examples of some of the physiological andmorphological characteristics of the variety HOI002 includecharacteristics related to yield, maturity, and kernel quality, each ofwhich is specifically disclosed herein. The regenerable cells in suchtissue cultures will preferably be derived from embryos, meristematiccells, immature tassels, microspores, pollen, leaves, anthers, roots,root tips, silk, flowers, kernels, ears, cobs, husks, or stalks, or fromcallus or protoplasts derived from those tissues. Still further, thepresent invention provides corn plants regenerated from the tissuecultures of the invention, the plants having all the physiological andmorphological characteristics of variety HOI002.

In yet another aspect of the invention, processes are provided forproducing corn seeds or plants, which processes generally comprisecrossing a first parent corn plant with a second parent corn plant,wherein at least one of the first or second parent corn plants is aplant of the variety designated HOI002. These processes may be furtherexemplified as processes for preparing hybrid corn seed or plants,wherein a first inbred corn plant is crossed with a second corn plant ofa different, distinct variety to provide a hybrid that has, as one ofits parents, the inbred corn plant variety HOI002. In these processes,crossing will result in the production of seed. The seed productionoccurs regardless of whether the seed is collected or not.

In a preferred embodiment of the invention, the first step in “crossing”comprises planting, preferably in pollinating proximity, seeds of afirst and second parent corn plant, and preferably, seeds of a firstinbred corn plant and a second, distinct inbred corn plant. Where theplants are not in pollinating proximity, pollination can nevertheless beaccomplished by transferring a pollen or tassel bag from one plant tothe other as described below.

A second step comprises cultivating or growing the seeds of said firstand second parent corn plants into plants that bear flowers. Corn bearsboth male flowers (tassels) and female flowers (silks) in separateanatomical structures on the same plant.

A third step comprises preventing self-pollination of the plants, i.e.,preventing the silks of a plant from being fertilized by any plant ofthe same variety, including the same plant. This is preferably done byemasculating the male flowers of the first or second parent corn plant,(i.e., treating or manipulating the flowers so as to prevent pollenproduction, in order to produce an emasculated parent corn plant),Self-incompatibility systems are also used in some hybrid crops for thesame purpose. Self-incompatible plants still shed viable pollen and canpollinate plants of other varieties but are incapable of pollinatingthemselves or other plants of the same variety.

A fourth step comprises allowing cross-pollination to occur between thefirst and second parent corn plants. When the plants are not inpollinating proximity, this is done by placing a bag, usually paper orglassine, over the tassels of the first plant and another bag over thesilks of the incipient ear on the second plant. The bags are left inplace for at least 24 hours. Since pollen is viable for less than 24hours, this assures that the silks are not pollinated from other pollensources, that any stray pollen on the tassels of the first plant isdead, and that the only pollen transferred comes from the first plant.The pollen bag over the tassel of the first plant is then shakenvigorously to enhance release of pollen from the tassels, and the shootbag is removed from the silks of the incipient ear on the second plant.Finally, the pollen bag is removed from the tassel of the first plantand is placed over the silks of the incipient ear of the second plant,shaken again and left in place. Yet another step comprises harvestingthe seeds from at least one of the parent corn plants. The harvestedseed can be grown to produce a corn plant or hybrid corn plant.

The present invention also provides corn seed and plants produced by aprocess that comprises crossing a first parent corn plant with a secondparent corn plant, wherein at least one of the first or second parentcorn plants is a plant of the variety designated HOI002. In oneembodiment of the invention, corn seed and plants produced by theprocess are first generation (F₁) hybrid corn seed and plants producedby crossing an inbred in accordance with the invention with another,distinct inbred. The present invention further contemplates seed of anF₁ hybrid corn plant. Therefore, certain exemplary embodiments of theinvention provide an F₁ hybrid corn plant and seed thereof.

In still yet another aspect of the invention, the genetic complement ofthe corn plant variety designated HOI002 is provided. The phrase“genetic complement” is used to refer to the aggregate of nucleotidesequences, the expression of which sequences defines the phenotype of,in the present case, a corn plant, or a cell or tissue of that plant. Agenetic complement thus represents the genetic make up of an inbredcell, tissue or plant, and a hybrid genetic complement represents thegenetic make up of a hybrid cell, tissue or plant. The invention thusprovides corn plant cells that have a genetic complement in accordancewith the inbred corn plant cells disclosed herein, and plants, seeds anddiploid plants containing such cells.

In still yet another aspect, the present invention provides hybridgenetic complements, as represented by corn plant cells, tissues,plants, and seeds, formed by the combination of a haploid geneticcomplement of an inbred corn plant of the invention with a haploidgenetic complement of a second corn plant, preferably, another, distinctinbred corn plant. In another aspect, the present invention provides acorn plant regenerated from a tissue culture that comprises a hybridgenetic complement of this invention.

In still yet another aspect, the present invention provides a method ofproducing an inbred corn plant derived from the corn variety HOI002, themethod comprising the steps of: (a) preparing a progeny plant derivedfrom corn variety HOI002, wherein said preparing comprises crossing aplant of the corn variety HOI002 with a second corn plant, and wherein asample of the seed of corn variety HOI002 has been deposited under ATCCAccession No. PTA-3788; (b) crossing the progeny plant with itself or asecond plant to produce a seed of a progeny plant of a subsequentgeneration; (c) growing a progeny plant of a subsequent generation fromsaid seed of a progeny plant of a subsequent generation and crossing theprogeny plant of a subsequent generation with itself or a second plant;and (d) repeating steps (c) and (d) for an additional 2-10 generations,including at least about 3, 5, 7, 9, 10 or more generations, to producean inbred corn plant derived from the corn variety HOI002. In themethod, it may be desirable to select particular plants resulting fromstep (c) for continued crossing according to steps (b) and (c). Byselecting plants having one or more desirable traits, an inbred cornplant derived from the corn variety HOI002 is obtained that possessessome of the desirable traits of corn variety HOI002 as well potentiallyother selected traits.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions of PlantCharacteristics

Barren Plants: Plants that are barren, i.e., lack an ear with grain, orhave an ear with only a few scattered kernels.

Cg: Colletotrichum graminicola rating. Rating times 10 is approximatelyequal to percent total plant infection.

CLN: Corn Lethal Necrosis (combination of Maize Chlorotic Mottle Virusand Maize Dwarf Mosaic virus) rating: numerical ratings are based on aseverity scale where 1=most resistant to 9=susceptible.

Cn: Corynebacterium nebraskense rating. Rating times 10 is approximatelyequal to percent total plant infection.

Cz: Cercospora zeae-maydis rating. Rating times 10 is approximatelyequal to percent total plant infection.

Dgg: Diatraea grandiosella girdling rating (values are percent plantsgirdled and stalk lodged).

Dropped Ears: Ears that have fallen from the plant to the ground.

Dsp: Diabrotica species root ratings (1=least affected to 9=severepruning).

Ear-Attitude: The attitude or position of the ear at harvest scored asupright, horizontal, or pendant.

Ear-Cob Color: The color of the cob, scored as white, pink, red, orbrown.

Ear-Cob Diameter: The average diameter of the cob measured at themidpoint.

Ear-Cob Strength: A measure of mechanical strength of the cobs tobreakage, scored as strong or weak.

Ear-Diameter: The average diameter of the ear at its midpoint.

Ear-Dry Husk Color: The color of the husks at harvest scored as buff,red, or purple.

Ear-Fresh Husk Color: The color of the husks 1 to 2 weeks afterpollination scored as green, red, or purple.

Ear-Husk Bract: The length of an average husk leaf scored as short,medium, or long.

Ear-Husk Cover: The average distance from the tip of the ear to the tipof the husks. Minimum value no less than zero.

Ear-Husk Opening: An evaluation of husk tightness at harvest scored astight, intermediate, or open.

Ear-Length: The average length of the ear.

Ear-Number Per Stalk: The average number of ears per plant.

Ear-Shank Internodes: The average number of internodes on the ear shank.

Ear-Shank Length: The average length of the ear shank.

Ear-Shelling Percent: The average of the shelled grain weight divided bythe sum of the shelled grain weight and cob weight for a single ear.

Ear-Silk Color: The color of the silk observed 2 to 3 days after silkemergence scored as green-yellow, yellow, pink, red, or purple.

Ear-Taper (Shape): The taper or shape of the ear scored as conical,semi-conical, or cylindrical.

Ear-Weight: The average weight of an ear.

Early Stand: The percent of plants that emerge from the ground asdetermined in the early spring.

ER: Ear rot rating (values approximate percent ear rotted).

Final Stand Count: The number of plants just prior to harvest.

GDUs: Growing degree units are calculated herein by the Barger Method,where the heat units for a 24-h period are calculated as GDUs=[(Maximumdaily temperature+Minimum daily temperature)/2]−50. The highest maximumdaily temperature used is 86° F. and the lowest minimum temperature usedis 50° F.

GDUs to Shed: The number of growing degree units (GDUs) or heat unitsrequired for an inbred line or hybrid to have approximately 50% of theplants shedding pollen as measured from time of planting. GDUs to shedis determined by summing the individual GDU daily values from plantingdate to the date of 50% pollen shed.

GDUs to Silk: The number of growing degree units for an inbred line orhybrid to have approximately 50% of the plants with silk emergence asmeasured from time of planting. GDUs to silk is determined by summingthe individual GDU daily values from planting date to the date of 50%silking.

Hc2: Helminthosporium carbonum race 2 rating. Rating times 10 isapproximately equal to percent total plant infection.

Hc3: Helminthosporium carbonum race 3 rating. Rating times 10 isapproximately equal to percent total plant infection.

Hm: Helminthosporium maydis race 0 rating. Rating times 10 isapproximately equal to percent total plant infection.

Ht1: Helminthosporium turcicum race 1 rating. Rating times 10 isapproximately equal to percent total plant infection.

Ht2: Helminthosporium turcicum race 2 rating. Rating times 10 isapproximately equal to percent total plant infection.

HtG: Chlorotic-lesion type resistance. += indicates the presence of Htchlorotic-lesion type resistance; −= indicates absence of Htchlorotic-lesion type resistance; and +/−= indicates segregation of Htchlorotic-lesion type resistance. Rating times 10 is approximately equalto percent total plant infection.

Kernel-Aleurone Color: The color of the aleurone scored as white, pink,tan, brown, bronze, red, purple, pale purple, colorless, or variegated.

Kernel-Cap Color: The color of the kernel cap observed at dry stage,scored as white, lemon-yellow, yellow, or orange.

Kernel-Endosperm Color: The color of the endosperm scored as white, paleyellow, or yellow.

Kernel-Endosperm Type: The type of endosperm scored as normal, waxy, oropaque.

Kernel-Grade: The percent of kernels that are classified as rounds.

Kernel-Length: The average distance from the cap of the kernel to thepedicel.

Kernel-Number Per Row: The average number of kernels in a single row.

Kernel-Pericarp Color: The color of the pericarp scored as colorless,red-white crown, tan, bronze, brown, light red, cherry red, orvariegated.

Kernel-Row Direction: The direction of the kernel rows on the ear scoredas straight, slightly curved, spiral, or indistinct (scattered).

Kernel-Row Number: The average number of rows of kernels on a singleear.

Kernel-Side Color: The color of the kernel side observed at the drystage, scored as white, pale yellow, yellow, orange, red, or brown.

Kernel-Thickness: The distance across the narrow side of the kernel.

Kernel-Type: The type of kernel scored as dent, flint, or intermediate.

Kernel-Weight: The average weight of a predetermined number of kernels.

Kernel-Width: The distance across the flat side of the kernel.

Kz: Kabatiella zeae rating. Rating times 10 is approximately equal topercent total plant infection.

Leaf-Angle: Angle of the upper leaves to the stalk scored as upright (0to 30 degrees), intermediate (30 to 60 degrees), or lax (60 to 90degrees).

Leaf-Color: The color of the leaves 1 to 2 weeks after pollinationscored as light green, medium green, dark green, or very dark green.

Leaf-Length: The average length of the primary ear leaf.

Leaf-Longitudinal Creases: A rating of the number of longitudinalcreases on the leaf surface 1 to 2 weeks after pollination. Creases arescored as absent, few, or many.

Leaf-Marginal Waves: A rating of the waviness of the leaf margin 1 to 2weeks after pollination. Rated as none, few, or many.

Leaf-Number: The average number of leaves of a mature plant. Countingbegins with the cotyledonary leaf and ends with the flag leaf.

Leaf-Sheath Anthocyanin: A rating of the level of anthocyanin in theleaf sheath 1 to 2 weeks after pollination, scored as absent,basal-weak, basal-strong, weak or strong.

Leaf-Sheath Pubescence: A rating of the pubescence of the leaf sheath.Ratings are taken 1 to 2 weeks after pollination and scored as light,medium, or heavy.

Leaf-Width: The average width of the primary ear leaf measured at itswidest point.

LSS: Late season standability (values times 10 approximate percentplants lodged in disease evaluation plots).

Moisture: The moisture of the grain at harvest.

On1: Ostrinia nubilalis 1st brood rating (1=resistant to 9=susceptible).

On2: Ostrinia nubilalis 2nd brood rating (1=resistant to 9=susceptible).

Relative Maturity: A maturity rating based on regression analysis. Theregression analysis is developed by utilizing check hybrids and theirpreviously established day rating versus actual harvest moistures.Harvest moisture on the hybrid in question is determined and thatmoisture value is inserted into the regression equation to yield arelative maturity.

Root Lodging: Root lodging is the percentage of plants that root lodge.A plant is counted as root lodged if a portion of the plant leans fromthe vertical axis by approximately 30 degrees or more.

Seedling Color: Color of leaves at the 6 to 8 leaf stage.

Seedling Height: Plant height at the 6 to 8 leaf stage.

Seedling Vigor: A visual rating of the amount of vegetative growth on a1 to 9 scale, where 1 equals best. The score is taken when the averageentry in a trial is at the fifth leaf stage.

Selection Index: The selection index gives a single measure of hybrid'sworth based on information from multiple traits. One of the traits thatis almost always included is yield. Traits may be weighted according tothe level of importance assigned to them.

Sr: Sphacelotheca reiliana rating is actual percent infection.

Stalk-Anthocyanin: A rating of the amount of anthocyanin pigmentation inthe stalk. The stalk is rated 1 to 2 weeks after pollination as absent,basal-weak, basal-strong, weak, or strong.

Stalk-Brace Root Color: The color of the brace roots observed 1 to 2weeks after pollination as green, red, or purple.

Stalk-Diameter: The average diameter of the lowest visible internode ofthe stalk.

Stalk-Ear Height: The average height of the ear measured from the groundto the point of attachment of the ear shank of the top developed ear tothe stalk.

Stalk-Internode Direction: The direction of the stalk internode observedafter pollination as straight or zigzag.

Stalk-Internode Length: The average length of the internode above theprimary ear.

Stalk Lodging: The percentage of plants that did stalk lodge. Plants arecounted as stalk lodged if the plant is broken over or off below theear.

Stalk-Nodes With Brace Roots: The average number of nodes having braceroots per plant.

Stalk-Plant Height: The average height of the plant as measured from thesoil to the tip of the tassel.

Stalk-Tillers: The percent of plants that have tillers. A tiller isdefined as a secondary shoot that has developed as a tassel capable ofshedding pollen.

Staygreen: Staygreen is a measure of general plant health near the timeof black layer formation (physiological maturity). It is usuallyrecorded at the time the ear husks of most entries within a trial haveturned a mature color. Scoring is on a 1 to 9 basis where 1 equals best.

STR: Stalk rot rating (values represent severity rating of 1=25% ofinoculated internode rotted to 9=entire stalk rotted and collapsed).

SVC: Southeastern Virus Complex (combination of Maize Chlorotic DwarfVirus and Maize Dwarf Mosaic Virus) rating; numerical ratings are basedon a severity scale where 1=most resistant to 9=susceptible (1988reactions are largely Maize Dwarf Mosaic Virus reactions).

Tassel-Anther Color: The color of the anthers at 50% pollen shed scoredas green-yellow, yellow, pink, red, or purple.

Tassel-Attitude: The attitude of the tassel after pollination scored asopen or compact.

Tassel-Branch Angle: The angle of an average tassel branch to the mainstem of the tassel scored as upright (less than 30 degrees),intermediate (30 to 45 degrees), or lax (greater than 45 degrees).

Tassel-Branch Number: The average number of primary tassel branches.

Tassel-Glume Band: The closed anthocyanin band at the base of the glumescored as present or absent.

Tassel-Glume Color: The color of the glumes at 50% shed scored as green,red, or purple.

Tassel-Length: The length of the tassel measured from the base of thebottom tassel branch to the tassel tip.

Tassel-Peduncle Length: The average length of the tassel peduncle,measured from the base of the flag leaf to the base of the bottom tasselbranch.

Tassel-Pollen Shed: A visual rating of pollen shed determined by tappingthe tassel and observing the pollen flow of approximately five plantsper entry. Rated on a 1 to 9 scale where 9=sterile, 1=most pollen.

Tassel-Spike Length: The length of the spike measured from the base ofthe top tassel branch to the tassel tip.

Test Weight: Weight of the grain in pounds for a given volume (bushel)adjusted to 15.5% moisture.

Yield: Yield of grain at harvest adjusted to 15.5% moisture.

II. Other Definitions

Allele: Any of one or more alternative forms of a gene locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny back to one of the parents, for example, a first generationhybrid (F₁) with one of the parental genotypes of the F₁ hybrid.

Chromatography: A technique wherein a mixture of dissolved substancesare bound to a solid support followed by passing a column of fluidacross the solid support and varying the composition of the fluid. Thecomponents of the mixture are separated by selective elution.

Crossing: The pollination of a female flower of a corn plant, therebyresulting in the production of seed from the flower.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Electrophoresis: A process by which particles suspended in a fluid or agel matrix are moved under the action of an electrical field, andthereby separated according to their charge and molecular weight. Thismethod of separation is well known to those skilled in the art and istypically applied to separating various forms of enzymes and of DNAfragments produced by restriction endonucleases.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a chemical agent or a cytoplasmic or nuclear geneticfactor conferring male sterility.

Enzymes: Molecules that can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two plants.

Genetic Complement: An aggregate of nucleotide sequences, the expressionof which sequences defines the phenotype in corn plants, or componentsof plants including cells or tissue.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Isozymes: Detectable variants of an enzyme, the variants catalyzing thesame reaction(s) but differing from each other, e.g., in primarystructure and/or electrophoretic mobility. The differences betweenisozymes are under single gene, codominant control. Consequently,electrophoretic separation to produce band patterns can be equated todifferent alleles at the DNA level. Structural differences that do notalter charge cannot be detected by this method.

Isozyme typing profile: A profile of band patterns of isozymes separatedby electrophoresis that can be equated to different alleles at the DNAlevel.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Metaxenia: An effect exerted on the phenotype of the embryo andassociated diploid tissues of a seed by the genotype contributed by thepollen.

HOI002: The corn plant variety from which seeds having ATCC AccessionNo. PTA-3788 were obtained, as well as plants grown from those seeds.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Genetic loci that contribute, at least inpart, certain numerically representable traits that are usuallycontinuously distributed.

Regeneration: The development of a plant from tissue culture.

SSR profile: A profile of simple sequence repeats used as geneticmarkers and scored by gel electrophoresis following PCR™ amplificationusing flanking oligonucleotide primers.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Single Locus Converted (Conversion) Plant: Plants that are developed bya plant breeding technique called backcrossing, wherein essentially allof the desired morphological and physiological characteristics of aninbred are recovered in addition to the characteristics conferred by thesingle locus transferred into the inbred via the backcrossing technique.A single locus may comprise one gene, or in the case of transgenicplants, one or more transgenes integrated into the host genome at asingle site (locus).

Substantially equivalent: The modifier “substantially equivalent” asused with respect to a first numerical value, such as that associatedwith a quantitative trait, for example, is used to include thosenumerical values that, when compared to the first numerical value, donot show statistical differences of their respective means.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic sequence that has been introduced into the nuclearor chloroplast genome of a maize plant by a genetic transformationtechnique.

Xenia: An effect exerted on the phenotype of the endosperm of a seed bythe genotype contributed by the pollen.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

II. Inbred Corn Plant HOI002

In accordance with one aspect of the present invention, there isprovided a novel inbred corn plant variety designated HOI002. Cornvariety HOI002 shows uniformity and stability within the limits ofenvironmental influence for the traits described hereinafter in Table 1.HOI002 has been self-pollinated and ear-rowed a sufficient number ofgenerations with careful attention paid to uniformity of plant type toensure homozygosity and phenotypic stability. No variant traits havebeen observed or are expected in HOI002.

Inbred corn plants can be reproduced by planting the seeds of the inbredcorn plant HOI002, growing the resulting corn plants underself-pollinating or sib-pollinating conditions with adequate isolationusing standard techniques well known to an artisan skilled in theagricultural arts. Seeds can be harvested from such a plant usingstandard, well known procedures.

A. Phenotypic Description

In accordance with another aspect of the present invention, there isprovided a corn plant having the physiological and morphologicalcharacteristics of corn plant HOI002. A description of the physiologicaland morphological characteristics of corn plant HOI002 is given inTable 1. TABLE 1 Physiological and Morphological Traits for HOI002MATURITY: Days Heat Units From emergence to 50% of plants in silk: 801524 From emergence to 50% of plants in pollen 79 1496 Heat Units: =$\frac{\begin{matrix}\lbrack {{{Max}.\quad{Temp}.\quad( {\leq {86{^\circ}\quad{F.}}} )} +}  \\{ {{Min}.\quad{Temp}\quad.\quad.\quad.\quad( {\geq {50{^\circ}\quad{F.}}} )} \rbrack - 50}\end{matrix}}{2}$ PLANT: Plant Height (to tassel tip): 206.0 cm EarHeight (to base of top ear): 112.0 cm Average number of Tillers: 0Average Number of Ears per Stalk: 1 Anthocyanin of Brace Roots: AbsentLEAF: Width of Ear Node Leaf: 9.0 cm Length of Ear Node Leaf: 72.0 cmNumber of leaves above top ear: 5 Leaf Angle (from 2nd Leaf above ear atanthesis to Stalk above leaf): 27° Leaf Color: Medium Green - MunsellColor Code 5 GY 4/4 Leaf Sheath Pubescence (Rate on scale from 1 = noneto 9 = like peach fuzz): 2 Marginal Waves (Rate on scale from 1 = noneto 9 = many): 4 Longitudinal Creases (Rate on scale from 1 = none to 9 =many): 5 TASSEL: Number of Lateral Branches: 4 Branch Angle from CentralSpike: 15° Tassel Length (from top leaf collar to tassel top): 39.0 cmPollen Shed (Rate on scale from 0 = male sterile to 9 = heavy shed): 6Anther Color: Green-Yellow - Munsell Color Code 2.5 GY 8/10 Glume Color:Green - Munsell Color Code 5 GY 5/8 Bar Glumes: Absent EAR (UnhuskedData) Silk Color (3 days after emergence): Light Green - Munsell ColorCode 2.5 GY 8/6 Fresh Husk Color (25 days after 50% silking): LightGreen - Munsell Color Code 2.5 GY 7/8 Dry Husk Color (65 days after 50%silking): Buff - Munsell Color Code 7.5 YR 7/4 Husk Extension: Short(ears exposed) EAR: (Husked Ear Data) Ear Length: 12.0 cm Ear Diameterat mid-point: 42.0 mm Ear Weight: 66.0 gm Number of Kernel Rows: 18Kernel Rows: Distinct Shank Length: 10.0 cm Ear Taper: Average KERNEL:(Dried) Kernel Length: 10.0 mm Kernel Width: 7.0 mm Kernel Thickness:4.0 mm Round Kernels (Shape Grade): Aleurone Color Pattern: HomozygousCOB: Cob Diameter at Mid-Point: 3.1*These are typical values. Values may vary due to environment. Othervalues that are substantially equivalent are also within the scope ofthe invention.

B. Deposit Information

A representative deposit of 2500 seeds of the inbred corn varietydesignated HOI002 has been made with the American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va. on Oct. 19,2001. Those deposited seeds have been assigned ATCC Accession No.PTA-3788. The deposit was made in accordance with the terms andprovisions of the Budapest Treaty relating to deposit of microorganismsand was made for a term of at least thirty (30) years and at least five(05) years after the most recent request for the finishing of a sampleof the deposit is received by the depository, or for the effective termof the patent, whichever is longer, and will be replaced if it becomesnon-viable during that period.

IV. Single Locus Conversions

When the term inbred corn plant is used in the context of the presentinvention, this also includes any single locus conversions of thatinbred. The term single locus converted plant as used herein refers tothose corn plants that are developed by a plant breeding techniquecalled backcrossing, wherein essentially all of the desiredmorphological and physiological characteristics of an inbred arerecovered in addition to the single locus transferred into the inbredvia the backcrossing technique. Backcrossing methods can be used withthe present invention to improve or introduce a characteristic into theinbred. The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to one of the parental corn plants forthat inbred. The parental corn plant that contributes the locus or locifor the desired characteristic is termed the nonrecurrent or donorparent. This terminology refers to the fact that the nonrecurrent parentis used one time in the backcross protocol and therefore does not recur.The parental corn plant to which the locus or loci from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman et al., 1995;Fehr, 1987; Sprague and Dudley, 1988). In a typical backcross protocol,the original inbred of interest (recurrent parent) is crossed to asecond inbred (nonrecurrent parent) that carries the single locus ofinterest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a corn plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred locus from the nonrecurrent parent. The backcross processmay be accelerated by the use of genetic markers, such as SSR, RFLP, SNPor AFLP markers, to identify plants with the greatest genetic complementfrom the recurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalinbred. To accomplish this, a single locus of the recurrent inbred ismodified or substituted with the desired locus from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original inbred. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single locus traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single locus traits may or may not betransgenic; examples of these traits include, but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability, andyield enhancement. These genes are generally inherited through thenucleus, but may be inherited through the cytoplasm. Some knownexceptions to this are genes for male sterility, some of which areinherited cytoplasmically, but still act as single locus traits. Anumber of exemplary single locus traits are described in, for example,PCT Application WO 95/06128, the disclosure of which is specificallyincorporated herein by reference.

Examples of genes conferring male sterility include those disclosed inU.S. Pat. No. 3,861,709, U.S. Pat. No. 3,710,511, U.S. Pat. No.4,654,465, U.S. Pat. No. 5,625,132, and U.S. Pat. No. 4,727,219, each ofthe disclosures of which are specifically incorporated herein byreference in their entirety. A particularly useful type of malesterility gene is one that can be induced by exposure to a chemicalagent, for example, a herbicide (U.S. patent Ser. No. 08/927,368, filedSep. 11, 1997, the disclosure of which is specifically incorporatedherein by reference in its entirety). Both inducible and non-induciblemale sterility genes can increase the efficiency with which hybrids aremade, in that they eliminate the need to physically emasculate the cornplant used as a female in a given cross.

Where one desires to employ male-sterility systems with a corn plant inaccordance with the invention, it may be beneficial to also utilize oneor more male-fertility restorer genes. For example, where cytoplasmicmale sterility (CMS) is used, hybrid seed production requires threeinbred lines: (1) a cytoplasmically male-sterile line having a CMScytoplasm; (2) a fertile inbred with normal cytoplasm, which is isogenicwith the CMS line for nuclear genes (“maintainer line”); and (3) adistinct, fertile inbred with normal cytoplasm, carrying a fertilityrestoring gene (“restorer” line). The CMS line is propagated bypollination with the maintainer line, with all of the progeny being malesterile, as the CMS cytoplasm is derived from the female parent. Thesemale sterile plants can then be efficiently employed as the femaleparent in hybrid crosses with the restorer line, without the need forphysical emasculation of the male reproductive parts of the femaleparent.

The presence of a male-fertility restorer gene results in the productionof fully fertile F₁ hybrid progeny. If no restorer gene is present inthe male parent, male-sterile hybrids are obtained. Such hybrids areuseful where the vegetative tissue of the corn plant is utilized, e.g.,for silage, but in most cases, the seeds will be deemed the mostvaluable portion of the crop, so fertility of the hybrids in these cropsmust be restored. Therefore, one aspect of the current inventionconcerns the inbred corn plant HOI0012 comprising a single gene capableof restoring male fertility in an otherwise male-sterile inbred orhybrid plant. Examples of male-sterility genes and correspondingrestorers that could be employed with the inbred of the invention arewell known to those of skill in the art of plant breeding and aredisclosed in, for instance, U.S. Pat. No. 5,530,191; U.S. Pat. No.5,689,041; U.S. Pat. No. 5,741,684; and U.S. Pat. No. 5,684,242, thedisclosures of which are each specifically incorporated herein byreference in their entirety.

Direct selection may be applied where a single locus acts as a dominanttrait. An example of a dominant trait is the herbicide resistance trait.For this selection process, the progeny of the initial cross are sprayedwith the herbicide prior to the backcrossing. The spraying eliminatesany plants that do not have the desired herbicide resistancecharacteristic, and only those plants that have the herbicide resistancegene are used in the subsequent backcross. This process is then repeatedfor all additional backcross generations.

Many useful single locus traits are those that are introduced by genetictransformation techniques. Methods for the genetic transformation ofmaize are known to those of skill in the art. For example, methods thathave been described for the genetic transformation of maize includeelectroporation (U.S. Pat. No. 5,384,253), electrotransformation (U.S.Pat. No. 5,371,003), microprojectile bombardment (U.S. Pat. No.5,550,318; U.S. Pat. No. 5,736,369, U.S. Pat. No. 5,538,880; and PCTPublication WO 95/06128), Agrobacterium-mediated transformation (U.S.Pat. No. 5,591,616 and E.P. Publication EP672752), direct DNA uptaketransformation of protoplasts (Omirulleh et al., 1993) and siliconcarbide fiber-mediated transformation (U.S. Pat. No. 5,302,532 and U.S.Pat. No. 5,464,765).

A type of single locus trait that can be introduced by genetictransformation (U.S. Pat. No. 5,554,798) and has particular utility is agene that confers resistance to the herbicide glyphosate. Glyphosateinhibits the action of the enzyme EPSPS, which is active in thebiosynthetic pathway of aromatic amino acids. Inhibition of this enzymeleads to starvation for the amino acids phenylalanine, tyrosine, andtryptophan and secondary metabolites derived therefrom. Mutants of thisenzyme are available that are resistant to glyphosate. For example, U.S.Pat. No. 4,535,060 describes the isolation of EPSPS mutations thatconfer glyphosate resistance upon organisms having the Salmonellatyphimurium gene for EPSPS, aroA. A mutant EPSPS gene having similarmutations has also been cloned from Zea mays. The mutant gene encodes aprotein with amino acid changes at residues 102 and 106 (PCT PublicationWO 97/04103). When a plant comprises such a gene, a herbicide resistantphenotype results.

Plants having inherited a transgene comprising a mutated EPSPS gene may,therefore, be directly treated with the herbicide glyphosate without theresult of significant damage to the plant. This phenotype providesfarmers with the benefit of controlling weed growth in a field of plantshaving the herbicide resistance trait by application of the broadspectrum herbicide glyphosate. For example, one could apply theherbicide ROUNDUP™, a commercial formulation of glyphosate manufacturedand sold by the Monsanto Company, over the top in fields whereglyphosate resistant corn plants are grown. The herbicide applicationrates may typically range from 4 ounces of ROUNDUP™ to 256 ouncesROUNDUP™ per acre. More preferably, about 16 ounces to about 64 ouncesper acre of ROUNDUP™ may be applied to the field. However, theapplication rate may be increased or decreased as needed, based on theabundance and/or type of weeds being treated. Additionally, depending onthe location of the field and weather conditions, which will influenceweed growth and the type of weed infestation, it may be desirable toconduct further glyphosate treatments. The second glyphosate applicationwill also typically comprise an application rate of about 16 ounces toabout 64 ounces of ROUNDUP™ per acre treated. Again, the treatment ratemay be adjusted based on field conditions. Such methods of applicationof herbicides to agricultural crops are well known in the art and aresummarized in general in Anderson (1983).

Alternatively, more than one single locus trait may be introgressed intoan elite inbred by the method of backcross conversion. A selectablemarker gene and a gene encoding a protein that confers a trait ofinterest may be simultaneously introduced into a maize plant as a resultof genetic transformation. Usually one or more introduced genes willintegrate into a single chromosome site in the host cell's genome. Forexample, a selectable marker gene encoding phosphinothricin acetyltransferase (PPT) (e.g., a bar gene) and conferring resistance to theactive ingredient in some herbicides by inhibiting glutamine synthetase,and a gene encoding an endotoxin from Bacillus thuringiensis (Bt) andconferring resistance to particular classes of insects, e.g.,lepidopteran insects, in particular the European Corn Borer, may besimultaneously introduced into a host genome. Furthermore, through theprocess of backcross conversion more than one transgenic trait may betransferred into an elite inbred.

The waxy characteristic is an example of a recessive trait. In thisexample, the progeny resulting from the first backcross generation (BC1)must be grown and selfed. A test is then run on the selfed seed from theBC1 plant to determine which BC1 plants carried the recessive gene forthe waxy trait. In other recessive traits additional progeny testing,for example growing additional generations such as the BC1S1, may berequired to determine which plants carry the recessive gene.

V. Origin and Breeding History of an Exemplary Single Locus ConvertedPlant

Methods for the preparation of single gene converted plants are known tothose of skill in the art. An example of such a single gene convertedplant is 85DGD1 MLms, which is a single locus conversion of the cornline 85DGD1 to cytoplasmic male sterility. The methods used to convertthis line were described in U.S. Pat. No. 6,175,063, the disclosure ofwhich is incorporated herein in the entirety, and are set forth hereinbelow. As described in U.S. Pat. No. 6,175,063, 85DGD1 MLms was derivedusing backcross methods. In particular, the line designated 85DGD1 wasused as the recurrent parent and MLms, a germplasm source carrying MLcytoplasmic sterility, was used as the nonrecurrent parent. The breedinghistory of the single locus converted inbred 85DGD1 MLms can besummarized as follows: Hawaii Nurseries Planting Made up S-O: Female row585 male Date Apr. 02, 1992 row 500 Hawaii Nurseries Planting S-O wasgrown and plants were Date Jul. 15, 1992 backcrossed times 85DGD1 (rows444 ′ 443) Hawaii Nurseries Planting Bulked seed of the BC1 was grownDate Nov. 18, 1992 and backcrossed times 85DGD1 (rows V3-27 ′ V3-26)Hawaii Nurseries Planting Bulked seed of the BC2 was grown and Date Apr.02, 1993 backcrossed times 85DGD1 (rows 37 ′ 36) Hawaii NurseriesPlanting Bulked seed of the BC3 was grown and Date Jul. 14, 1993backcrossed times 85DGD1 (rows 99 ′ 98) Hawaii Nurseries Planting Bulkedseed of BC4 was grown and Date Oct. 28, 1993 backcrossed times 85DGD1(rows KS-63 ′ KS-62) Summer 1994 A single ear of the BC5 was grown andbackcrossed times 85DGD1 (MC94-822 ′ MC94-822-7) Winter 1994 Bulked seedof the BC6 was grown and backcrossed times 85DGD1 (3Q-1 ′ 3Q-2) Summer1995 Seed of the BC7 was bulked and named 85DGD1 MLms.VI. Tissue Cultures and In Vitro Regeneration of Corn Plants

A further aspect of the invention relates to tissue cultures of the cornplant designated HOI002. As used herein, the term “tissue culture”indicates a composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant. Exemplary types of tissue cultures are protoplasts, calli andplant cells that are intact in plants or parts of plants, such asembryos, pollen, flowers, kernels, ears, cobs, leaves, husks, stalks,roots, root tips, anthers, silk, and the like. In a preferredembodiment, the tissue culture comprises embryos, protoplasts,meristematic cells, pollen, leaves or anthers derived from immaturetissues of these plant parts. Means for preparing and maintaining planttissue cultures are well known in the art (U.S. Pat. No. 5,538,880; andU.S. Pat. No. 5,550,318, each incorporated herein by reference in theirentirety). By way of example, a tissue culture comprising organs such astassels or anthers has been used to produce regenerated plants (U.S.Pat. No. 5,445,961 and U.S. Pat. No. 5,322,789; the disclosures of whichare incorporated herein by reference).

VII. Tassel/Anther Culture

Tassels contain anthers that in turn enclose microspores. Microsporesdevelop into pollen. For anther/microspore culture, if tassels are theplant composition, they are preferably selected at a stage when themicrospores are uninucleate, that is, include only one, rather than 2 or3 nuclei. Methods to determine the correct stage are well known to thoseskilled in the art and include mitramycin fluorescent staining (Pace etal., 1987), trypan blue (preferred) and acetocarmine squashing. Themid-uninucleate microspore stage has been found to be the developmentalstage most responsive to the subsequent methods disclosed to ultimatelyproduce plants.

Although microspore-containing plant organs such as tassels cangenerally be pretreated at any cold temperature below about 25° C., arange of 4 to 25° C. is preferred, and a range of 8 to 14° C. isparticularly preferred. Although other temperatures yield embryoids andregenerated plants, cold temperatures produce optimum response ratescompared to pretreatment at temperatures outside the preferred range.Response rate is measured as either the number of embryoids or thenumber of regenerated plants per number of microspores initiated inculture. Exemplary methods of microspore culture are disclosed in, forexample, U.S. Pat. No. 5,322,789 and U.S. Pat. No. 5,445,961, thedisclosures of which are specifically incorporated herein by reference.

Although not required, when tassels are employed as the plant organ, itis generally preferred to sterilize their surface. Following surfacesterilization of the tassels, for example, with a solution of calciumhypochloride, the anthers are removed from about 70 to 150 spikelets(small portions of the tassels) and placed in a preculture orpretreatment medium. Larger or smaller amounts can be used depending onthe number of anthers.

When one elects to employ tassels directly, tassels are preferablypretreated at a cold temperature for a predefined time, preferably at10° C. for about 4 days. After pretreatment of a whole tassel at a coldtemperature, dissected anthers are further pretreated in an environmentthat diverts microspores from their developmental pathway. The functionof the preculture medium is to switch the developmental program from oneof pollen development to that of embryoid/callus development. Anembodiment of such an environment in the form of a preculture mediumincludes a sugar alcohol, for example mannitol or sorbitol, inositol orthe like. An exemplary synergistic combination is the use of mannitol ata temperature of about 10° C. for a period ranging from about 10 to 14days. In a preferred embodiment, 3 ml of 0.3 M mannitol combined with 50mg/l of ascorbic acid, silver nitrate, and colchicine is used forincubation of anthers at 10° C. for between 10 and 14 days. Anotherembodiment is to substitute sorbitol for mannitol. The colchicineproduces chromosome doubling at this early stage. The chromosomedoubling agent is preferably only present at the preculture stage.

It is believed that the mannitol or other similar carbon structure orenvironmental stress induces starvation and functions to forcemicrospores to focus their energies on entering developmental stages.The cells are unable to use, for example, mannitol as a carbon source atthis stage. It is believed that these treatments confuse the cellscausing them to develop as embryoids and plants from microspores.Dramatic increases in development from these haploid cells, as high as25 embryoids in 10⁴ microspores, have resulted from using these methods.

In embodiments where microspores are obtained from anthers, microsporescan be released from the anthers into an isolation medium following themannitol preculture step. One method of release is by disruption of theanthers, for example, by chopping the anthers into pieces with a sharpinstrument, such as a razor blade, scalpel, or Waring blender. Theresulting mixture of released microspores, anther fragments, andisolation medium are then passed through a filter to separatemicrospores from anther wall fragments. An embodiment of a filter is amesh, more specifically, a nylon mesh of about 112 mm pore size. Thefiltrate that results from filtering the microspore-containing solutionis preferably relatively free of anther fragments, cell walls, and otherdebris.

In a preferred embodiment, isolation of microspores is accomplished at atemperature below about 25° C. and preferably, at a temperature of lessthan about 15° C. Preferably, the isolation media, dispersing tool(e.g., razor blade), funnels, centrifuge tubes, and dispersing container(e.g., petri dish) are all maintained at the reduced temperature duringisolation. The use of a precooled dispersing tool to isolate maizemicrospores has been reported (Gaillard et al., 1991).

Where appropriate and desired, the anther filtrate is then washedseveral times in isolation medium. The purpose of the washing andcentrifugation is to eliminate any toxic compounds that are contained inthe non-microspore part of the filtrate and are created by the choppingprocess. The centrifugation is usually done at decreasing spin speeds,for example, 1000, 750, and finally 500 rpms. The result of theforegoing steps is the preparation of a relatively pure tissue culturesuspension of microspores that are relatively free of debris and antherremnants.

To isolate microspores, an isolation media is preferred. An isolationmedia is used to separate microspores from the anther walls whilemaintaining their viability and embryogenic potential. An illustrativeembodiment of an isolation media includes a 6% sucrose or maltosesolution combined with an antioxidant such as 50 mg/l of ascorbic acid,0.1 mg/l biotin, and 400 mg/l of proline, combined with 10 mg/l ofnicotinic acid and 0.5 mg/l AgNO₃. In another embodiment, the biotin andproline are omitted.

An isolation media preferably has a higher antioxidant level where it isused to isolate microspores from a donor plant (a plant from which aplant composition containing a microspore is obtained) that is fieldgrown in contrast to greenhouse grown. A preferred level of ascorbicacid in an isolation medium is from about 50 mg/l to about 125 mg/l and,more preferably, from about 50 mg/l to about 100 mg/l.

One can find particular benefit in employing a support for themicrospores during culturing and subculturing. Any support thatmaintains the cells near the surface can be used. The microsporesuspension is layered onto a support, for example by pipetting. Thereare several types of supports that are suitable and are within the scopeof the invention. An illustrative embodiment of a solid support is aTRANSWELL® culture dish. Another embodiment of a solid support fordevelopment of the microspores is a bilayer plate wherein liquid mediais on top of a solid base. Other embodiments include a mesh or amillipore filter. Preferably, a solid support is a nylon mesh in theshape of a raft. A raft is defined as an approximately circular supportmaterial that is capable of floating slightly above the bottom of atissue culture vessel, for example, a petri dish, of about a 60 or 100mm size, although any other laboratory tissue culture vessel willsuffice. In an illustrative embodiment, a raft is about 55 mm indiameter.

Culturing isolated microspores on a solid support, for example, on a 10mm pore nylon raft floating on 2.2 ml of medium in a 60 mm petri dish,prevents microspores from sinking into the liquid medium and thusavoiding low oxygen tension. These types of cell supports enable theserial transfer of the nylon raft with its associatedmicrospore/embryoids ultimately to full strength medium containingactivated charcoal and solidified with, for example, GELRITE™(solidifying agent). The charcoal is believed to absorb toxic wastes andintermediaries. The solid medium allows embryoids to mature.

The liquid medium passes through the mesh while the microspores areretained and supported at the medium-air interface. The surface tensionof the liquid medium in the petri dish causes the raft to float. Theliquid is able to pass through the mesh; consequently, the microsporesstay on top. The mesh remains on top of the total volume of liquidmedium. An advantage of the raft is to permit diffusion of nutrients tothe microspores. Use of a raft also permits transfer of the microsporesfrom dish to dish during subsequent subculture with minimal loss,disruption, or disturbance of the induced embryoids that are developing.The rafts represent an advantage over the multi-welled TRANSWELL®(plates, which are commercially available from COSTAR, in that thecommercial plates are expensive. Another disadvantage of these plates isthat to achieve the serial transfer of microspores to subsequent media,the membrane support with cells must be peeled off the insert in thewells. This procedure does not produce as good a yield nor as efficienttransfers, as when a mesh is used as a vehicle for cell transfer.

The culture vessels can be further defined as either (1) a bilayer 60 mmpetri plate wherein the bottom 2 ml of medium are solidified with 0.7%agarose overlaid with 1 mm of liquid containing the microspores; (2) anylon mesh raft wherein a wafer of nylon is floated on 1.2 ml of mediumand 1 ml of isolated microspores is pipetted on top; or (3) TRANSWELL®plates wherein isolated microspores are pipetted onto membrane insertsthat support the microspores at the surface of 2 ml of medium.

After the microspores have been isolated, they are cultured in a lowstrength anther culture medium until about the 50 cell stage when theyare subcultured onto an embryoid/callus maturation medium. Medium isdefined at this stage as any combination of nutrients that permit themicrospores to develop into embryoids or callus. Many examples ofsuitable embryoid/callus promoting media are well known to those skilledin the art. These media will typically comprise mineral salts, a carbonsource, vitamins, and growth regulators. A solidifying agent isoptional. A preferred embodiment of such a media is referred to as “Dmedium,” which typically includes 6N1 salts, AgNO₃ and sucrose ormaltose.

In an illustrative embodiment, 1 ml of isolated microspores are pipettedonto a 10 mm nylon raft and the raft is floated on 1.2 ml of medium “D,”containing sucrose or preferably maltose. Both calli and embryoids candevelop. Calli are undifferentiated aggregates of cells. Type I is arelatively compact, organized, and slow growing callus. Type II is asoft, friable, and fast-growing one. Embryoids are aggregates exhibitingsome embryo-like structures. The embryoids are preferred for subsequentsteps to regenerating plants. Culture medium “D” is an embodiment ofmedium that follows the isolation medium and replaces it. Medium “D”promotes growth to an embryoid/callus. This medium comprises 6N1 saltsat ⅛ the strength of a basic stock solution (major components) and minorcomponents, plus 12% sucrose, or preferably 12% maltose, 0.1 mg/l B1,0.5 mg/l nicotinic acid, 400 mg/l proline and 0.5 mg/l silver nitrate.Silver nitrate is believed to act as an inhibitor to the action ofethylene. Multi-cellular structures of approximately 50 cells eachgenerally arise during a period of 12 days to 3 weeks. Serial transferafter a two week incubation period is preferred.

After the petri dish has been incubated for an appropriate period oftime, preferably two weeks in the dark at a predefined temperature, araft bearing the dividing microspores is transferred serially to solidbased media that promote embryo maturation. In an illustrativeembodiment, the incubation temperature is 30° C. and the mesh raftsupporting the embryoids is transferred to a 100 mm petri dishcontaining the 6N1-TGR-4P medium, an “anther culture medium.” Thismedium contains 6N1 salts, supplemented with 0.1 mg/l TIBA, 12% sugar(sucrose, maltose, or a combination thereof), 0.5% activated charcoal,400 mg/l proline, 0.5 mg/l B, 0.5 mg/l nicotinic acid, and 0.2 percentGELRITE™ (solidifying agent) and is capable of promoting the maturationof the embryoids. Higher quality embryoids, that is, embryoids thatexhibit more organized development, such as better shoot meristemformation without precocious germination, were typically obtained withthe transfer to full strength medium compared to those resulting fromcontinuous culture using only, for example, the isolated microsporeculture (IMC) Medium “D.” The maturation process permits the pollenembryoids to develop further in route toward the eventual regenerationof plants. Serial transfer occurs to full strength solidified 6N1 mediumusing either the nylon raft, the TRANSWELL® membrane, or bilayer plates,each one requiring the movement of developing embryoids to permitfurther development into physiologically more mature structures. In anespecially preferred embodiment, microspores are isolated in anisolation media comprising about 6% maltose, cultured for about twoweeks in an embryoid/calli induction medium comprising about 12% maltoseand then transferred to a solid medium comprising about 12% sucrose.

At the point of transfer of the raft, after about two weeks ofincubation, embryoids exist on a nylon support. The purpose oftransferring the raft with the embryoids to a solidified medium afterthe incubation is to facilitate embryo maturation. Mature embryoids atthis point are selected by visual inspection indicated by zygoticembryo-like dimensions and structures and are transferred to the shootinitiation medium. It is preferred that shoots develop before roots, orthat shoots and roots develop concurrently. If roots develop beforeshoots, plant regeneration can be impaired. To produce solidified media,the bottom of a petri dish of approximately 100 mm is covered with about30 ml of 0.2% GELRITE™ solidified medium. A sequence of regenerationmedia are used for whole plant formation from the embryoids.

During the regeneration process, individual embryoids are induced toform plantlets. The number of different media in the sequence can varydepending on the specific protocol used. Finally, a rooting medium isused as a prelude to transplanting to soil. When plantlets reach aheight of about 5 cm, they are then transferred to pots for furthergrowth into flowering plants in a greenhouse by methods well known tothose skilled in the art.

Plants have been produced from isolated microspore cultures by themethods disclosed herein, including self-pollinated plants. The rate ofembryoid induction was much higher with the synergistic preculturetreatment consisting of a combination of stress factors, including acarbon source that can be capable of inducing starvation, a coldtemperature, and colchicine, than has previously been reported. Anillustrative embodiment of the synergistic combination of treatmentsleading to the dramatically improved response rate compared to priormethods, is a temperature of about 10° C., mannitol as a carbon source,and 0.05% colchicine.

The inclusion of ascorbic acid, an anti-oxidant, in the isolation mediumis preferred for maintaining good microspore viability. However, thereseems to be no advantage to including mineral salts in the isolationmedium. The osmotic potential of the isolation medium was maintainedoptimally with about 6% sucrose, although a range of 2% to 12% is withinthe scope of this invention.

In an embodiment of the embryoid/callus organizing media, mineral saltsconcentration in IMC Culture Media “D” is (⅛×), the concentration thatis used also in anther culture medium. The 6N1 salts major componentshave been modified to remove ammonium nitrogen. Osmotic potential in theculture medium is maintained with about 12% sucrose and about 400 mg/lproline. Silver nitrate (0.5 mg/l) was included in the medium to modifyethylene activity. The preculture media is further characterized byhaving a pH of about 5.7 to 6.0. Silver nitrate and vitamins do notappear to be crucial to this medium but do improve the efficiency of theresponse.

Whole anther cultures can also be used in the production ofmonocotyledonous plants from a plant culture system. There are somebasic similarities of anther culture methods and microspore culturemethods with regard to the media used. A difference from isolatedmicrospore cultures is that undisrupted anthers are cultured, so that asupport, e.g., a nylon mesh support, is not needed. The first step indeveloping the anther cultures is to incubate tassels at a coldtemperature. A cold temperature is defined as less than about 25° C.More specifically, the incubation of the tassels is preferably performedat about 10° C. A range of 8 to 14° C. is also within the scope of theinvention. The anthers are then dissected from the tassels, preferablyafter surface sterilization using forceps, and placed on solidifiedmedium. An example of such a medium is designated 6N1-TGR-P4.

The anthers are then treated with environmental conditions that arecombinations of stresses that are capable of diverting microspores fromgametogenesis to embryogenesis. It is believed that the stress effect ofsugar alcohols in the preculture medium, for example, mannitol, isproduced by inducing starvation at the predefined temperature. In oneembodiment, the incubation pretreatment is for about 14 days at 10° C.It was found that treating the anthers in addition with a carbonstructure, an illustrative embodiment being a sugar alcohol, preferablymannitol, produces dramatically higher anther culture response rates asmeasured by the number of eventually regenerated plants, than bytreatment with either cold treatment or mannitol alone. These resultsare particularly surprising in light of teachings that cold is betterthan mannitol for these purposes, and that warmer temperatures interactwith mannitol better.

To incubate the anthers, they are floated on a preculture medium thatdiverts the microspores from gametogenesis, preferably on a mannitolcarbon structure, more specifically, 0.3 M of mannitol plus 50 mg/l ofascorbic acid. Three milliliters is about the total amount in a dish,for example, a tissue culture dish, more specifically, a 60 mm petridish. Anthers are isolated from about 120 spikelets for one dish yieldsabout 360 anthers.

Chromosome doubling agents can be used in the preculture media foranther cultures. Several techniques for doubling chromosome number(Jensen, 1974; Wan et al., 1989) have been described. Colchicine is oneof the doubling agents. However, developmental abnormalities arisingfrom in vitro cloning are further enhanced by colchicine treatments, andprevious reports indicated that colchicine is toxic to microspores. Theaddition of colchicine in increasing concentrations during mannitolpretreatment prior to anther culture and microspore culture has achievedimproved percentages.

An illustrative embodiment of the combination of a chromosome doublingagent and preculture medium is one that contains colchicine. In aspecific embodiment, the colchicine level is preferably about 0.05%. Theanthers remain in the mannitol preculture medium with the additives forabout 10 days at 10° C. Anthers are then placed on maturation media, forexample, that designated 6N1-TGR-P4, for 3 to 6 weeks to induceembryoids. If the plants are to be regenerated from the embryoids, shootregeneration medium is employed, as in the isolated microspore proceduredescribed in the previous sections. Other regeneration media can be usedsequentially to complete regeneration of whole plants.

The anthers are then exposed to embryoid/callus promoting medium, forexample, that designated 6N1-TGR-P4, to obtain callus or embryoids. Theembryoids are recognized visually by identification of embryonic-likestructures. At this stage, the embryoids are transferred progressivelythrough a series of regeneration media. In an illustrative embodiment,the shoot initiation medium comprises BAP (6-benzyl-amino-purine) andNAA (naphthalene acetic acid). Regeneration protocols for isolatedmicrospore cultures and anther cultures are similar.

VIII. Additional Tissue Cultures and Regeneration

The present invention contemplates a corn plant regenerated from atissue culture of the inbred maize plant HOI002, or of a hybrid maizeplant produced by crossing HOI002. As is well known in the art, tissueculture of corn can be used for the in vitro regeneration of a cornplant. By way of example, a process of tissue culturing and regenerationof corn is described in European Patent Application 0 160 390, thedisclosure of which is incorporated herein by reference. Corn tissueculture procedures are also described in Green and Rhodes (1982) andDuncan et al. (1985). The study by Duncan et al. (1985) indicates that97 percent of cultured plants produced calli capable of regeneratingplants. Subsequent studies have shown that both inbreds and hybridsproduced 91% regenerable calli that produced plants.

Other studies indicate that non-traditional tissues are capable ofproducing somatic embryogenesis and plant regeneration (Songstad et al.,1988; Rao et al., 1986; Conger et al., 1987; the disclosures of whichare incorporated herein by reference). Regenerable cultures, includingType I and Type II cultures, may be initiated from immature embryosusing methods described in, for example, PCT Application WO 95/06128,the disclosure of which is incorporated herein by reference in itsentirety.

Briefly, by way of example, to regenerate a plant of this invention,cells are selected following growth in culture. Where employed, culturedcells are preferably grown either on solid supports or in the form ofliquid suspensions as set forth above. In either instance, nutrients areprovided to the cells in the form of media, and environmental conditionsare controlled. There are many types of tissue culture media comprisingamino acids, salts, sugars, hormones, and vitamins. Most of the mediaemployed to regenerate inbred and hybrid plants have some similarcomponents; the media differ in the composition and proportions of theiringredients depending on the particular application envisioned. Forexample, various cell types usually grow in more than one type of media,but exhibit different growth rates and different morphologies, dependingon the growth media. In some media, cells survive but do not divide.Various types of media suitable for culture of plant cells have beenpreviously described and discussed above.

An exemplary embodiment for culturing recipient corn cells in suspensioncultures includes using embryogenic cells in Type II (Armstrong andGreen, 1985; Gordon-Kamm et al., 1990) callus, selecting for small (10to 30 mm) isodiametric, cytoplasmically dense cells, growing the cellsin suspension cultures with hormone containing media, subculturing intoa progression of media to facilitate development of shoots and roots,and finally, hardening the plant and readying it metabolically forgrowth in soil.

Meristematic cells (i.e., plant cells capable of continual cell divisionand characterized by an undifferentiated cytological appearance,normally found at growing points or tissues in plants such as root tips,stem apices, lateral buds, etc.) can be cultured (U.S. Pat. No.5,736,369, the disclosure of which is specifically incorporated hereinby reference).

Embryogenic calli are produced essentially as described in PCTApplication WO 95/06128. Specifically, inbred plants or plants fromhybrids produced from crossing an inbred of the present invention withanother inbred are grown to flowering in a greenhouse. Explants from atleast one of the following F₁ tissues: the immature tassel tissue,intercalary meristems and leaf bases, apical meristems, immature earsand immature embryos are placed in an initiation medium that contain MSsalts, supplemented with thiamine, agar, and sucrose. Cultures areincubated in the dark at about 23° C. All culture manipulations andselections are performed with the aid of a dissecting microscope.

After about 5 to 7 days, cellular outgrowths are observed from thesurface of the explants. After about 7 to 21 days, the outgrowths aresubcultured by placing them into fresh medium of the same composition.Some of the intact immature embryo explants are placed on fresh medium.Several subcultures later (after about 2 to 3 months) enough material ispresent from explants for subdivision of these embryogenic calli intotwo or more pieces.

Callus pieces from different explants are not mixed. After furthergrowth and subculture (about 6 months after embryogenic callusinitiation), there are usually between 1 and 100 pieces derivedultimately from each selected explant. During this time of cultureexpansion, a characteristic embryogenic culture morphology develops as aresult of careful selection at each subculture. Any organized structuresresembling roots or root primordia are discarded. Material known fromexperience to lack the capacity for sustained growth is also discarded(translucent, watery, embryogenic structures). Structures with a firmconsistency resembling at least in part the scutelum of the in vivoembryo are selected.

The callus is maintained on agar-solidified MS or N6-type media. Apreferred hormone is 2,4-D. A second preferred hormone is dicamba.Visual selection of embryo-like structures is done to obtainsubcultures. Transfer of material other than that displaying embryogenicmorphology results in loss of the ability to recover whole plants fromthe callus.

Cell suspensions are prepared from the calli by selecting cellpopulations that appear homogeneous macroscopically. A portion of thefriable, rapidly growing embryogenic calli is inoculated into MS or N6Medium containing 2,4-D or dicamba. The calli in medium are incubated atabout 27° C. on a gyrotary shaker in the dark or in the presence of lowlight. The resultant suspension culture is transferred about once everythree to seven days, preferably every three to four days, by takingabout 5 to 10 ml of the culture and introducing this inoculum into freshmedium of the composition listed above (PCT Application WO 95/06128).

For regeneration of type I or type II callus, callus is transferred to asolidified culture medium that includes a lower concentration of 2,4-Dor other auxins than is present in culture medium used for callusmaintenance (PCT Application WO 95/06128, specifically incorporatedherein by reference). Other hormones that can be used in regenerationmedia include dicamba, NAA, ABA, BAP, and 2-NCA. Regeneration of plantsis completed by the transfer of mature and germinating embryos to ahormone-free medium, followed by the transfer of developed plantlets tosoil and growth to maturity. Plant regeneration is described in PCTApplication WO 95/06128.

Cells from the meristem or cells fated to contribute to the meristem ofa cereal plant embryo at the early proembryo, mid proembryo, lateproembryo, transitional or early coleoptilar stage may be cultured so asto produce a proliferation of shoots or multiple meristems from whichfertile plants may be regenerated. Alternatively, cells from themeristem or cells fated to contribute to the meristem of a cereal plantimmature ear or tassel may be cultured so as to produce a proliferationof shoots or multiple meristems from which fertile plants may beregenerated (U.S. Pat. No. 5,736,369).

Progeny of any generation are produced by taking pollen and selfing,backcrossing, or sibling crossing regenerated plants by methods wellknown to those skilled in the arts. Seeds are collected from theregenerated plants. Alternatively, progeny of any generation may beproduced by pollinating a regenerated plant with its own pollen orpollen of a second maize plant. Using the methods described herein,tissue cultures and immature or mature plant tissues may be used asrecipient cell cultures for the process of genetic transformation.

Ix. Processes of Preparing Corn Plants and the Corn Plants Produced bySuch Crosses

The present invention also provides a process of preparing a novel cornplant and a corn plant produced by such a process. In accordance withsuch a process, a first parent corn plant is crossed with a secondparent corn plant wherein at least one of the first and second cornplants is the inbred corn plant HOI002. An important aspect of thisprocess is that it can be used for the development of novel inbredlines. For example, the inbred corn plant HOI002 could be crossed to anysecond plant, and the resulting hybrid progeny each selfed for about 5to 7 or more generations, thereby providing a large number of distinct,pure-breeding inbred lines. These inbred lines could then be crossedwith other inbred or non-inbred lines and the resulting hybrid progenyanalyzed for beneficial characteristics. In this way, novel inbred linesconferring desirable characteristics could be identified.

In selecting a second plant to cross with HOI002 for the purpose ofdeveloping novel inbred lines, it will typically be desired choose thoseplants that either themselves exhibit one or more selected desirablecharacteristics or that exhibit the desired characteristic(s) when inhybrid combination. Examples of potentially desired characteristicsinclude greater yield, better stalks, better roots, resistance toinsecticides, herbicides, pests, and disease, tolerance to heat anddrought, reduced time to crop maturity, better agronomic quality, highernutritional value, and uniformity in germination times, standestablishment, growth rate, maturity, and fruit size. Alternatively, theinbred variety HOI002 may be crossed with a second, different inbredplant for the purpose of producing hybrid seed that is sold to farmersfor planting in commercial production fields. In this case, a secondinbred variety is selected that confers desirable characteristics whenin hybrid combination with the first inbred line.

Corn plants (Zea mays L.) can be crossed by either natural or mechanicaltechniques. Natural pollination occurs in corn when wind blows pollenfrom the tassels to the silks that protrude from the tops of therecipient ears. Mechanical pollination can be effected either bycontrolling the types of pollen that can blow onto the silks or bypollinating by hand.

In a preferred embodiment, crossing comprises the steps of:

-   -   (a) planting in pollinating proximity seeds of a first and a        second parent corn plant, and preferably, seeds of a first        inbred corn plant and a second, distinct inbred corn plant;    -   (b) cultivating or growing the seeds of the first and second        parent corn plants into plants that bear flowers;    -   (c) emasculating flowers of either the first or second parent        corn plant, i.e., treating the flowers so as to prevent pollen        production, or alternatively, using as the female parent a male        sterile plant, thereby providing an emasculated parent corn        plant;    -   (d) allowing natural cross-pollination to occur between the        first and second parent corn plants;    -   (e) harvesting seeds produced on the emasculated parent corn        plant; and, where desired,    -   (f) growing the harvested seed into a corn plant, preferably, a        hybrid corn plant.

Parental plants are typically planted in pollinating proximity to eachother by planting the parental plants in alternating rows, in blocks orin any other convenient planting pattern. Where the parental plantsdiffer in timing of sexual maturity, it may be desired to plant theslower maturing plant first, thereby ensuring the availability of pollenfrom the male parent during the time at which silks on the female parentare receptive to pollen. Plants of both parental parents are cultivatedand allowed to grow until the time of flowering. Advantageously, duringthis growth stage, plants are in general treated with fertilizer and/orother agricultural chemicals as considered appropriate by the grower.

At the time of flowering, in the event that plant HOI002 is employed asthe male parent, the tassels of the other parental plant are removedfrom all plants employed as the female parental plant to avoidself-pollination. The detasseling can be achieved manually but also canbe done by machine, if desired. Alternatively, when the female parentcorn plant comprises a cytoplasmic or nuclear gene conferring malesterility, detasseling may not be required. Additionally, a chemicalgametocide may be used to sterilize the male flowers of the femaleplant. In this case, the parent plants used as the male may either notbe treated with the chemical agent or may comprise a genetic factor thatcauses resistance to the emasculating effects of the chemical agent.Gametocides affect processes or cells involved in the development,maturation or release of pollen. Plants treated with such gametocidesare rendered male sterile, but typically remain female fertile. The useof chemical gametocides is described, for example, in U.S. Pat. No.4,936,904, the disclosure of which is specifically incorporated hereinby reference in its entirety. Furthermore, the use of Roundup herbicidein combination with glyphosate tolerant maize plants to produce malesterile corn plants is disclosed in U.S. patent application Ser. No.08/927,368 and PCT Publication WO 98/44140.

Following emasculation, the plants are then typically allowed tocontinue to grow and natural cross-pollination occurs as a result of theaction of wind, which is normal in the pollination of grasses, includingcorn. As a result of the emasculation of the female parent plant, allthe pollen from the male parent plant is available for pollinationbecause tassels, and thereby pollen bearing flowering parts, have beenpreviously removed from all plants of the inbred plant being used as thefemale in the hybridization. Of course, during this hybridizationprocedure, the parental varieties are grown such that they are isolatedfrom other corn fields to minimize or prevent any accidentalcontamination of pollen from foreign sources. These isolation techniquesare well within the skill of those skilled in this art.

Both parental inbred plants of corn may be allowed to continue to growuntil maturity or the male rows may be destroyed after flowering iscomplete. Only the ears from the female inbred parental plants areharvested to obtain seeds of a novel F₁ hybrid. The novel F₁ hybrid seedproduced can then be planted in a subsequent growing season incommercial fields or, alternatively, advanced in breeding protocols forpurposes of developing novel inbred lines.

Alternatively, in another embodiment of the invention, both first andsecond parent corn plants can come from the same inbred corn plant,i.e., from the inbred designated HOI002. Thus, any corn plant producedusing a process of the present invention and inbred corn plant HOI002,is contemplated by the current inventor. As used herein, crossing canmean selfing, backcrossing, crossing to another or the same inbred,crossing to populations, and the like. All corn plants produced usingthe inbred corn plant HOI002 as a parent are, therefore, within thescope of this invention.

The utility of the inbred plant HOI002 also extends to crosses withother species. Commonly, suitable species will be of the familyGraminaceae, and especially of the genera Zea, Tripsacum, Coix,Schlerachne, Polytoca, Chionachne, and Trilobachne, of the tribeMaydeae. Of these, Zea and Tripsacum, are most preferred. Potentiallysuitable for crosses with HOI002 can also be the various varieties ofgrain sorghum, Sorghum bicolor (L.) Moench.

A. Utilization of HOI002 as a Pollinator for Enhancing Kernel QualityTraits

One advantage of the variety of the invention is that it possessesmarkedly enhanced quality grain traits relative to typical priorvarieties. These traits can be conferred through crossing for theproduction of kernels and plants grown therefrom with enhanced qualitytraits. Such enhanced traits may include, for example, elevated oilcontent, elevated protein content, improved oil quality, enhancedoxidative stability of the oil, reduced polyunsaturated fatty acids inthe oil, elevated oleic acid content, improved nutritional quality ofthe protein and improved functional properties of the starch, as well asother traits. In particular embodiments of the invention, plantsobtained can comprise one or more enhanced quality traits selected fromthe group consisting of: oil content in excess of 6% of the seed drymatter, protein content in excess of 10% of the seed dry matter, oleicacid content in excess of 35% of the total fatty acids of the oil,lysine content in excess of 0.32% of the seed dry matter and tryptophancontent in excess of 0.08% of the seed dry matter. Such enhanced qualitytraits may be conferred in hybrid combination.

The variety HOI002 can also be used to confer enhanced quality graintraits by way of the xenia and/or metaxenia effect, in which qualitytraits of pollen affect the grain resulting from the pollinating. Onemethod for exploiting the effect involves planting in pollinatingproximity in a field corn seed of the variety HOI002 and corn seed of anagronomically elite variety. By allowing these plants to grow together,pollen of the variety HOI002 becomes available to pollinate the femaleflowers of the agronomically elite variety. The pollinating confersenhanced quality grain traits upon the kernels produced by thepollinating, but the yield of grain will otherwise approach thatnormally obtained using the agronomically elite “female” parent.Preferably, the agronomically elite plant is rendered male sterile toprevent self pollination. This can be achieved genetically, manually orchemically, as is described herein. It is preferable that theagronomically elite parent has a similar maturity date to the varietyHOI002 to ensure coincidence of the availability of pollen andreceptivity thereto. The resulting kernels can then be harvested fromthe plants.

B. F₁ Hybrid Corn Plant and Seed Production

Any time the inbred corn plant HOI002 is crossed with another,different, corn inbred, a first generation (F₁) corn hybrid plant isproduced. As such, an F₁ hybrid corn plant may be produced by crossingHOI002 with any second inbred maize plant. Therefore, any F₁hybrid cornplant or corn seed that is produced with HOI002 as a parent is part ofthe present invention.

The goal of the process of producing an F_(‘)hybrid is to manipulate thegenetic complement of corn to generate new combinations of genes thatinteract to yield new or improved traits (phenotypic characteristics). Aprocess of producing an F₁ hybrid typically begins with the productionof one or more inbred plants. Those plants are produced by repeatedcrossing of ancestrally related corn plants to try to combine certaingenes within the inbred plants.

Corn has a diploid phase, which means two conditions of a gene (twoalleles) occupy each locus (position on a chromosome). If the allelesare the same at a locus, there is said to be homozygosity. If they aredifferent, there is said to be heterozygosity. In a completely inbredplant, all loci are homozygous. Because many loci when homozygous aredeleterious to the plant, in particular leading to reduced vigor, lesskernels, weak and/or poor growth, production of inbred plants is anunpredictable and arduous process. Under some conditions, heterozygousadvantage at some loci effectively bars perpetuation of homozygosity.

Inbreeding requires sophisticated manipulation by human breeders. Evenin the extremely unlikely event inbreeding rather than crossbreedingoccurred in natural corn, achievement of complete inbreeding cannot beexpected in nature due to well known deleterious effects of homozygosityand the large number of generations the plant would have to breed inisolation. The reason for the breeder to create inbred plants is to havea known reservoir of genes whose gametic transmission is predictable.The development of inbred plants generally requires at least about 5 to7 generations of selfing. Inbred plants are then cross-bred in anattempt to develop improved F₁ hybrids. Hybrids are then screened andevaluated in small scale field trials. Typically, about 10 to 15phenotypic traits, selected for their potential commercial value, aremeasured.

When the inbred corn plant HOI002 is crossed with another inbred plantto yield a hybrid, the original inbred can serve as either the maternalor paternal plant. For many crosses, the outcome is the same regardlessof the assigned sex of the parental plants. However, there is often oneof the parental plants that is preferred as the maternal plant becauseof increased seed yield and production characteristics. Some plantsproduce tighter ear husks leading to more loss, for example due to rot.There can be delays in silk formation that deleteriously affect timingof the reproductive cycle for a pair of parental inbreds. Seed coatcharacteristics can be preferable in one plant. Pollen can be shedbetter by one plant. Other variables can also affect preferred sexualassignment of a particular cross. In the case of the instant inbred,HOI002 can be crossed as either the male or female parent.

C. F₁ Hybrid Comparisons

As mentioned above, hybrids are progressively eliminated followingdetailed evaluations of their phenotype, including formal comparisonswith other commercially successful hybrids. Strip trials are used tocompare the phenotypes of hybrids grown in as many environments aspossible. They are performed in many environments to assess overallperformance of the new hybrids and to select optimum growing conditions.Because the corn is grown in close proximity, environmental factors thataffect gene expression, such as moisture, temperature, sunlight, andpests, are minimized. For a decision to be made to commercialize ahybrid, it is not necessary that the hybrid be better than all otherhybrids. Rather, significant improvements must be shown in at least sometraits that would create improvements in some niches.

In one aspect of the invention, comparative data may be used to assesshybrids produced using HOI002 as one parent. In one embodiment of theinvention, the data may comprise grain quality trait measurements, forexample, analysis of percent endosperm by dry weight, percent embryo bydry weight, percent oil in the endosperm by dry weight, oil in theembryo by dry weight percent and oil in the kernel by dry weight.Alternatively, a per kernel analysis could be used, for example, bycomparing traits such as endosperm size, embryo size, endosperm+embryoweight, oil weight in endosperm, oil weight in the embryo and oil weightin the endosperm+embryo. Examples of hybrid comparisons carried out onHOI002 are presented below, in Tables 2-4. TABLE 2 Enhancement of oilcontent in three-way hybrids with HOI002 Grain Stalk Root Oil YieldMoisture Lodging Lodging Content Pedigree (Bu/A) (%) (%) (%) (%) LH310 ×RQ0084 172.5 20.5 4.6 4.5 5.8 (LH310 × HOI002) × 131.1 20.1 7.0 16.7 9.8RQ0084 LH310 × RQ0085 177.7 25.5 2.8 5.4 6.1 (LH310 × HOI001) × 156.523.7 7.3 4.5 8.9 RQ0085

TABLE 3 Comparison of conventional hybrids to a hybrid of HOI002 GrainStalk Root Oil Yield Moisture Lodging Lodging Content Pedigree (Bu/A)(%) (%) (%) (%) (LH227 × LH228) × 175.5 18.0 21.2 11.8 4.5 LH82 (LH227 ×LH228) × 172.7 17.1 14.8 12.8 4.0 LH172 (LH227 × LH228) × 192.5 16.8 6.07.0 3.9 LH176 (LH227 × LH228) × 173.7 17.8 9.3 17.0 9.8 HOI002

TABLE 4 Comparison of conventional hybrids to a hybrid of HOI002 GrainStalk Root Oil Yield Moisture Lodging Lodging Content Pedigree (Bu/A)(%) (%) (%) (%) (HC33 × LH198) × 127.5 18.1 13.5 0.0 4.2 LH59 (HC33 ×LH198) × 149.5 19.1 6.1 2.4 4.3 LH185 (HC33 × LH198) × 143.2 19.9 13.03.5 4.7 LH283 (HC33 × LH198) × 128.2 20.3 11.2 13.4 9.9 HOI002X. Genetic Compliments

The present invention provides a genetic complement of the inbred cornplant variety designated HOI002. Further provided by the invention is ahybrid genetic complement, wherein the complement is formed by thecombination of a haploid genetic complement from HOI002 and anotherhaploid genetic complement. Means for determining such a geneticcomplement are well-known in the art.

As used herein, the phrase “genetic complement” means an aggregate ofnucleotide sequences, the expression of which defines the phenotype of acorn plant or a cell or tissue of that plant. By way of example, a cornplant is genotyped to determine a representative sample of the inheritedmarkers it possesses. Markers are alleles at a single locus. They arepreferably inherited in codominant fashion so that the presence of bothalleles at a diploid locus is readily detectable, and they are free ofenvironmental variation, i.e., their heritability is 1. This genotypingis preferably performed on at least one generation of the descendantplant for which the numerical value of the quantitative trait or traitsof interest are also determined. The array of single locus genotypes isexpressed as a profile of marker alleles, two at each locus. The markerallelic composition of each locus can be either homozygous orheterozygous. Homozygosity is a condition where both alleles at a locusare characterized by the same nucleotide sequence or size of a repeatedsequence. Heterozygosity refers to different conditions of the gene at alocus. A preferred type of genetic marker for use with the invention issimple sequence repeats (SSRs), although potentially any other type ofgenetic marker could be used, for example, restriction fragment lengthpolymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs),single nucleotide polymorphisms (SNPs), and isozymes.

A genetic marker profile of an inbred may be predictive of the agronomictraits of a hybrid produced using that inbred. For example, if an inbredof known genetic marker profile and phenotype is crossed with a secondinbred of known genetic marker profile and phenotype it is possible topredict the phenotype of the F₁ hybrid based on the combined geneticmarker profiles of the parent inbreds. Methods for prediction of hybridperformance from genetic marker data is disclosed in U.S. Pat. No.5,492,547, the disclosure of which is specifically incorporated hereinby reference in its entirety. Such predictions may be made using anysuitable genetic marker, for example, SSRs, RFLPs, AFLPs, SNPs, orisozymes.

SSRs are genetic markers based on polymorphisms in repeated nucleotidesequences, such as microsatellites. A marker system based on SSRs can behighly informative in linkage analysis relative to other marker systemsin that multiple alleles may be present. Another advantage of this typeof marker is that, through use of flanking primers, detection of SSRscan be achieved, for example, by the polymerase chain reaction (PCR™ ),thereby eliminating the need for labor-intensive Southern hybridization.The PCR™ detection is done by use of two oligonucleotide primersflanking the polymorphic segment of repetitive DNA. Repeated cycles ofheat denaturation of the DNA followed by annealing of the primers totheir complementary sequences at low temperatures, and extension of theannealed primers with DNA polymerase, comprise the major part of themethodology. Following amplification, markers can be scored by gelelectrophoresis of the amplification products. Scoring of markergenotype is based on the size (number of base pairs) of the amplifiedsegment.

Another aspect of this invention is a plant genetic complementcharacterized by a genetic isozyme typing profile. Isozymes are forms ofproteins that are distinguishable, for example, on starch gelelectrophoresis, usually by charge and/or molecular weight. Thetechniques and nomenclature for isozyme analysis are described in, forexample, Stuber et al. (1988), which is incorporated by reference.

A standard set of loci can be used as a reference set. Comparativeanalysis of these loci is used to compare the purity of hybrid seeds, toassess the increased variability in hybrids compared to inbreds, and todetermine the identity of seeds, plants, and plant parts. In thisrespect, an isozyme reference set can be used to develop genotypic“fingerprints.”

The present invention also provides a hybrid genetic complement formedby the combination of a haploid genetic complement of the corn plantHOI002 with a haploid genetic complement of a second corn plant. Meansfor combining a haploid genetic complement from the foregoing inbredwith another haploid genetic complement can comprise any method forproducing a hybrid plant from HOI002. It is contemplated that such ahybrid genetic complement can be prepared using in vitro regeneration ofa tissue culture of a hybrid plant of this invention.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1-24. (canceled)
 25. The hybrid corn seed of claim 26, wherein crossingcomprises the steps of: (a) planting the seeds of first and secondinbred corn plants; (b) cultivating the seeds of said first and secondinbred corn plants into plants that bear flowers; (c) preventing selfpollination of at least one of the first or second inbred corn plant;(d) allowing cross-pollination to occur between the first and secondinbred corn plants; and (e) harvesting seeds on at least one of thefirst or second inbred corn plants, said seeds resulting from saidcross-pollination.
 26. A hybrid corn seed produced by crossing a plantof the corn variety HOI002 with a second corn plant , wherein a sampleof the seed of the corn variety HOI002 was deposited under ATCCAccession No. ______.
 27. A hybrid corn plant produced by growing theseed of claim
 26. 28. (canceled)
 29. A hybrid corn seed produced bycrossing a plant of the corn variety HOI002 with a second corn plant,wherein a sample of the seed of the corn variety HOI002 was depositedunder ATCC Accession No. ______, wherein the plant of corn varietyHOI002 is further defined as having a genome comprising a single locusconversion.
 30. The corn seed of claim 29, wherein the single locus wasstably inserted into a corn genome by transformation.
 31. The corn seedof claim 29, wherein the locus is selected from the group consisting ofa dominant allele and a recessive allele.
 32. The corn seed of claim 29,wherein the locus confers a trait selected from the group consisting ofherbicide tolerance, insect resistance, resistance to bacterial, fungal,nematode or viral disease, yield enhancement, waxy starch, improvednutritional quality, enhanced yield stability, male sterility andrestoration of male fertility. 33-34. (canceled)
 35. A hybrid corn plantproduced by growing the seed of claim 29.